AbstractMulticolor fluorescence induced by UV light is a sensitive and specific tool that may be used to provide information about the primary and secondary metabolism of plants by monitoring signals of the chlorophyll fluorescence (Chl‐F) and blue‐green fluorescence (BGF), respectively. We have followed the systemic infection of Nicotiana benthamiana plants with the Pepper mild mottle virus (PMMoV) by means of a multicolor fluorescence‐imaging system, to detect differences between two strains of PMMoV during the infection process and to establish a correlation between the virulence and changes induced in the host plant. Changes in both BGF and Chl‐F were monitored. BGF increased mainly in the abaxial side of the leaf during pathogenesis and the corresponding images showed a clear vein‐associated pattern in leaves of infected plants. HPLC analysis of leaf extracts was carried out to identify compounds emitting BGF, and determined that chlorogenic acid was one of the main contributors. BGF imaging was able to detect viral‐induced changes in asymptomatic (AS) leaves before detection of the virus itself. Chl‐F images confirmed our previous results of alterations in the photosynthetic apparatus of AS leaves from infected plants that were detected with other imaging techniques. Fluorescence ratios F440/F690 and F440/F740, which increase during pathogenesis, were excellent indicators of biotic stress.